The environmental element selenium (Se) is essential for human health in small amounts, but toxic at high levels. There is strong evidence that even small changes in Se status have multi-faceted effects on human health which include altered immune function and susceptibility to viral infections, biochemical stresses, cancer, and even diabetes. Many of the beneficial effects of Se can be attributed to selenoproteins; proteins containing Se in the form of the 21st amino acid selenocysteine (Sec). The diversity of substrates and biochemical pathways that selenoproteins act upon likely explains the multiple health effects associated with Se intake. Selenocysteine is incorporated into selenoproteins during translation by an unconventional mechanism involving Sec-tRNA decoding of UGA codons. This translational redefinition of UGA codons provides a unique mechanism by which selenoprotein expression may be controlled. Consequently, the regulation of selenoprotein synthesis is quite complex and likely involves control of selenocysteine incorporation efficiency and mRNA stability (due to nonsense mediated decay) in addition to regulation of transcription and translation initiation. Here we propose to systematically examine and clarify the molecular mechanisms of selenocysteine incorporation and control of selenoprotein synthesis using a combination of new deep-sequencing approaches and well established biochemical methods. Ribosome profiling (deep-sequencing of ribosome protected mRNA footprints) will be used to examine the location and density of ribosomes actively translating selenoprotein mRNAs in vivo (mouse tissues). From this information it will be possible to quantify both translation initiation and Sec incorporation efficiency in the context of controlled changes in diet or genetic backgrounds. Combined with measurements of mRNA abundance, this methodology provides an ideal tool to determine precisely how changing biological conditions affect selenoprotein synthesis. Preliminary data is presented which challenges conventional thinking about the mechanism of selenocysteine incorporation and how cis- and trans-acting factors regulate selenoprotein expression. In Aim 1, we propose to continue our studies of the mechanisms by which altered dietary selenium intake affects selenoprotein expression. In Aim 2, we propose to test hypothesis regarding the mechanism of Sec incorporation using genetically modified mice containing mutations in key cis- and trans-acting factors required for Sec incorporation. Finally, in Aim 3 we propose to examine the impact of altered selenoprotein expression on transcriptional and translational control of gene expression across the genome.